Project Summary Mammalian cells have evolved multiple non-overlapping mechanisms to ensure that DNA replication initiates from origins of replications once and only once in each division cycle; loss of control over these mechanisms induces genomic instability, an important driver of malignant transformation. Increasing evidence suggests that origin utilization and activation in higher eukaryotes is influenced by epigenetic factors, but exact mechanisms are largely undefined. Our long-term goals are to elucidate the underpinning mechanisms that control replication initiation in mammalian cells and to understand how perturbations of these mechanisms provokes genomic instability. The histone methyltransferase SET8 is emerging as a key regulator of replication initiation in mammalian cells through its mono-methyltransferase activity on histone H4K20. The cell cycle regulated enzyme is essential for origin licensing in G1 phase of the cell cycle, but is proteolytically degraded in S-phase; blocking this step triggers reiterative replication initiation within the same cell cycle or re-replication. Both SET8 and H4K20me, however, are also involved in transcriptional repression and in the repair of DNA double strand breaks (DSBs), but whether these seemingly independent activities play a role in replication initiation or re-replication is not known. Most importantly, little to nothing is known about the nature or characteristics of the re-replication products that accumulate in cells with defective SET8 degradation, nor is there information on where in the genome re-replication occurs or if certain genomic regions are more prone to re-replication induction. Our new results show that re-replication resulting from defective SET8 degradation is not a stochastic process, with few genomic sites exhibit large and significant copy number gains, reminiscent of genomic amplifications that are seen in cancer cells. Additional preliminary studies suggest that re-replication may originate from DNA double strand breaks (DSBs) that may spontaneously arise during replication, and requires the activity of genes involved both in transcriptional silencing and in DSB repair. Our innovative preliminary studies and experimental approaches are designed to thoroughly examine this alternative model of re-replication induction. In Aim 1, we will determine the magnitude (copy number gains) and genomic distribution of the re-replicated DNA in bulk and single cells with defective SET8 degradation and following the induction of DSBs at defined genomic sites. We will also test if these parameters vary in different cancer cell types and in cancer vs. non-cancer cells. In Aim 2, we will define the roles of histone H4K20 methylation, transcriptional silencing by the H4K20me reader L3MBTL1, and proteins involved in the repair of DSBs in effecting re-replication. The successful execution of the proposed aims promises to increase our understanding of the mechanisms regulating replication initiation in mammalian cells, and lead to a better understanding of how perturbations of these mechanisms provokes genomic instability.